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PubMed1980

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis.

Three recombinant DNA vectors carrying the beta-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the beta-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded beta-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However S. cerevisiae transformed with LAC4 cannot grow on lactose, probably because lactose does not enter the cell.

Cloning, Molecular↗

PubMed1987

Expression of the human alpha-galactosidase A in Escherichia coli K-12.

We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.

Cloning, Molecular↗

PubMed1987

A genomic clone containing the promoter for the gene encoding the human lysosomal enzyme, alpha-galactosidase A.

We have isolated and characterized a human genomic clone for a lysosomal enzyme gene. The start point of transcription was identified using primer extension of poly(A)+ mRNA. This genomic clone is specific for human alpha-galactosidase A, and it includes sequences for the promoter, complete signal peptide, first exon, and part of the first intron. Direct and inverted repeat elements of 10, 11, 16, 19, and 22 nucleotides (nt) flank the promoter site. A (GA)n repeat element of approx. 60 nt with strong homology to similar elements identified in several species is located upstream from the promoter. A GGGCGG site specific for DNA-binding protein Sp1 is located near a CAAT box, and the CCGCCC inverted repeat of the Sp1 binding sequence is located by the TATA box. The sequence immediately flanking the ATG start codon of the human alpha-galactosidase A is highly homologous to sequences flanking the ATG start codons of the other human lysosomal hydrolases for which sequence information is available (beta-glucocerebrosidase, cathepsin B, cathepsin D, and beta-hexosaminidase alpha chain), but not for any of the other 133 human signal peptides examined. Our analysis also reveals that conversion of the propeptide to the mature enzyme involves cleavage of a C-terminal rather than an N-terminal fragment. This information about the normal alpha-galactosidase A gene will be useful for comparison to data obtained from patients with Fabry disease, who are characterized by a deficiency of this enzyme. This is the first genomic clone described to date for any lysosomal enzyme, and it establishes a reference for future analyses of the molecular events that mediate the expression of this important class of enzymes.

Amino Acid Sequence↗

PubMed1990

A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons.

A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons. To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted. The E. coli beta-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region. When introduced into the peripheral nervous system, this virus established latent infections and stably expressed beta-galactosidase in primary sensory neurons. Expression of beta-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus. Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.

Acute Disease↗

PubMed1988

In vitro activation of neuraminidase in the beta-galactosidase-neuraminidase-protective protein complex by cathepsin C.

Neuraminidase can be activated by incubation of crude glycoprotein fractions at acidic pH for 90 minutes at physiological temperature. This activation is inhibited by leupeptin. Incubation of the purified neuraminidase-beta-galactosidase-protective protein complex under the same conditions used for crude glycoprotein fractions did not lead to enhanced neuraminidase activity, but incubation in the presence of exogenous Cathepsin C at 4 degrees C resulted in marked enhancement of neuraminidase activity. This activation was again inhibited by leupeptin. Cathepsin D treatment resulted in destruction of neuraminidase under the same conditions and this effect was again inhibited by leupeptin. beta-galactosidase in crude glycoprotein fractions and in the complex was resistant to both Cathepsin C and D, while homogeneous beta-galactosidase was inactivated by these enzymes. We suggest that in vitro activation of neuraminidase may mimic the in vivo intralysosomal conversion of the neuraminidase precursor into the mature form of the enzyme.

Cathepsin C↗

PubMed1988

Site-directed mutagenic replacement of glu-461 with gln in beta-galactosidase (E. coli): evidence that glu-461 is important for activity.

Glutamic acid 461 of beta-galactosidase (E. coli) was replaced by gln using site-directed mutagenesis. Kinetic studies on the purified Q461-beta-galactosidase showed that it had less than 0.4% of the wild-type activity (with ONPG as substrate), confirming other studies which have suggested that the negative charge on glu-461 is important for activity. The Km values did not increase, indicating that binding of the substrate was not decreased by this change. Thermal denaturation studies showed Q461-beta-galactosidase to be somewhat more susceptible to heat denaturation than the wild-type enzyme.

Escherichia coli↗

PubMed1988

Electrophoretic study of alpha-D-galactosidases from seeds of Glycine soja and Vigna radiata possessing erythroagglutinating activity.

Polyacrylamide gel electrophoresis in an acidic buffer system was used to study the electrophoretic behaviour of two forms of alpha-D-galactosidase from seeds of soy bean (Glycine soja) and mung bean (Vigna radiata). The interaction of the enzymes with saccharides was monitored by affinity electrophoresis; for the preparation of affinity gels, water-soluble O-glycosyl polyacrylamide copolymers and polysaccharides were used. alpha-D-Galactosidases from both sources interact with immobilized alpha-D-galactosyl residues. On the basis of the results of affinity electrophoresis performed in the presence of various free sugars, dissociation constants for the complexes between alpha-D-galactosidase and free sugars were calculated.

Chromatography, Affinity↗

PubMed1985

Studies on lectins. LVIII. Sugar-binding properties, as determined by affinity electrophoresis, of alpha-D-galactosidases from Vicia faba seeds possessing erythroagglutinating activity.

The interaction of alpha-D-galactosidases from Vicia faba seeds with saccharides was studied by means of affinity electrophoresis on polyacrylamide gel in an acidic buffer system. For the preparation of affinity gels, water-soluble O-glycosyl polyacrylamide copolymers and polysaccharides were used. alpha-D-Galactosidases interact with immobilized O-alpha-D-galactosyl residues and glycogen, but no interaction was observed with immobilized O-alpha-D-mannosyl residues. On the basis of the results of affinity electrophoresis performed in the presence of various free sugars, dissociation constants of the various alpha-D-galactosidase-free sugar complexes were calculated.

Agglutination↗

PubMed1983

Structure and function of the yeast URA3 gene. Differentially regulated expression of hybrid beta-galactosidase from overlapping coding sequences in yeast.

Expression of the URA3 gene of Saccharomyces cerevisiae was studied by analysis of URA3-lacZ gene fusions constructed in vitro. Synthesis of hybrid beta-galactosidase by fusions in frame with the coding sequence for orotidine-5'-phosphate decarboxylase (OMPdecarboxylase) was found to be normally regulated even when only 11 nucleotides of URA3 coding sequence remained, indicating that all transcription initiation and regulatory sites are present at the beginning of the URA3 gene. An upstream initiator codon that begins a short overlapping coding sequence in another reading frame was also found to be active in producing hybrid beta-galactosidase. However this beta-galactosidase synthesis showed little or no regulation. Nuclease protection experiments revealed numerous species of URA3 mRNA. The regulation of these is consistent with the idea that the URA3 protein and the overlapping peptide are translated from differentially regulated mRNAs of different lengths.

Base Sequence↗

PubMed1989

Histochemical detection of alpha-D-galactosidase with 5-Br-4-Cl-3-indoxyl alpha-D-galactoside.

5-Br-4-Cl-3-indoxyl alpha-D-galactoside was used as a new substrate in azoindoxyl, indigogenic, and tetrazolium procedures for the detection of alpha-D-galactosidase in the light microscope. Compared with the simultaneous azo-dye methods using 1-naphthyl or 6-Br-2-naphthyl alpha-D-galactoside as substrates and hexazotized pararosaniline for simultaneous coupling, primarily the azoindoxyl method with hexazotized pararosaniline and the indigogenic technique proved to be superior. The azoindoxyl reaction is recommended for the localization of alpha-D-galactosidase in lysosomes when freeze-dried celloidin-coated cryostat sections are used: the indigogenic procedure should be employed for the detection of the total activity of the enzyme in combination with the technique of semipermeable membranes. The tetrazolium reaction delivered high amounts of formazan as the final reaction product; however, its localization was less precise than with the azoindoxyl and indigogenic methods. Conspicious species differences were found; except for the small intestine of suckling mice the highest activities of alpha-D-galactosidase were present in different immature and mature rat organs when compared with mouse, hamster, guinea-pig, marmoset monkey, and human tissues.

Aging↗

PubMed1986

beta-D-galactosidase activity in streptococci of serological group B.

Group B streptococci isolated from humans differed significantly in beta-D-galactosidase-activity from those of bovine mastitis. This could be demonstrated in a relatively simple and rapid test using a fluorogenic 4-methylumbelliferyl-beta-D-galactoside conjugate. Only 10 (12%) of 82 group B streptococcal cultures from human produced beta-D-galactosidase. On the other hand, 74 (96%) of 77 "bovine" cultures formed this enzyme. Thus, beta-D-galactosidase activity could be used as an additional marker for the differentiation between group B streptococci of human and bovine origin.

Acetylglucosaminidase↗

PubMed1983

Determination of beta-galactosidase activity in the intestinal tract of mice by ion-exchange high-performance liquid chromatography using epsilon-N-1-(1-deoxylactulosyl)-L-lysine as substrate.

epsilon-N-1-(1-Deoxylactulosyl)-L-lysine was synthesized and used as a substrate to assay beta-galactosidase activity. epsilon-N-1-(1-Deoxylactulosyl)-L-lysine and its degradation product epsilon-N-1-(1-deoxyfructosyl)-L-lysine were detected by high-voltage paper electrophoresis and ion-exchange high-performance liquid chromatography. The beta-galactosidase activity in different parts of the intestinal tract of germ-free and control mice was determined and compared with a beta-galactosidase activity which degrades lactose at pH 8.5 and 5.0 and which corresponded with bacterial and host enzymatic activities, respectively.

Amino Acids↗

PubMed1982

Inactivation of beta-Galactosidase by iodination of tyrosine-253.

Beta-Galactosidase is rapidly inactivated by iodination catalyzed by lactoperoxidase but is not inactivated in the presence of the substrate analogue, isopropyl beta-D-thiogalactoside (IPTG). Enzyme activity is lost upon the incorporation of 1 mol of iodine per mol of monomer, without dissociation of the tetrameric structure. Tryptic digests of beta-galactosidase iodinated with 125I in the presence and absence of IPTG were separated by high-performance liquid chromatography and were compared. One fraction was found to be more highly labeled in the digest from the inactivated protein. After isolation of the peptide, amino acid analysis indicated it to be Asp-Tyr-Leu-Arg, residues 252-255. Thus, Tyr-253 is the most reactive tyrosine in beta-galactosidase. This suggests that the conformation of this region of the protein may be altered by binding of IPTG to make Tyr-253 less accessible to iodination. Alternatively, Tyr-253 could be an active-site residue.

Amino Acid Sequence↗

PubMed1977

Purified human liver acid beta-D-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide.

Our studies with purified human liver acid beta-D-galactosidases (EC 3.2.1.23) indicate that 4-methylumbelliferyl beta-D-galactosidase and G(M1)-ganglioside beta-D-galactosidase activities are identical with lactosylceramidase II activity. Evidence for this includes co-purification of all enzyme activities by affinity chromatography to yield a single band on polyacrylamide-gel electrophoresis and coincident elution from Sepharose 6B of all three enzyme activities.

Cerebrosides↗

PubMed1983

Isolation and characterization of an endo-beta-galactosidase from Bacteroides fragilis.

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.

Bacteroides fragilis↗

PubMed1984

Glycosidases induced in Aspergillus tamarii. Mycelial alpha-D-galactosidases.

Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I.

Aspergillus↗

PubMed1984

Glycosidases induced in Aspergillus tamarii. Secreted alpha-D-galactosidase and beta-D-mannanase.

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.

Aspergillus↗

PubMed1978

Reduction of beta-galactosidase in the ketotic Chinese hamster kidney.

Kidneys from normal, diabetic-nonketotic and ketotic Chinese hamsters were homogenized, fractionated and assayed for beta-glucosidase, and beta-galactosidase activities. The kidneys of the ketotic animals were enlarged but the protein content in each subcellular fraction was similar in all three groups of animals. beta-Glucosidase was found chiefly in the soluble fraction and no difference was observed in these animals. beta-Galactosidase was distributed in both cytoplasmic and particulate fractions; difference in the specific activity of beta-galactosidase between control and ketotic animals was found in nuclear, lysosomal-mitochondrial, microsomal and soluble fractions.

Acidosis↗